CN106520964A - Double-recognition quantitative detection method for microRNA - Google Patents

Abstract

The invention discloses an upconversion fluorescence energy resonance transfer (UC‑LRET) technology combined with different hybridization and ligation double recognition processes, so as to realize single-step, homogeneous and highly sensitive detection of microRNA. It is mainly used in microRNA expression profile analysis, microRNA clinical diagnosis, microRNA research and detection, and microRNA-related drug research and screening. The present invention has the following advantages: (1) There are few operation steps, and it belongs to "one-step" detection. (2) It has good stability, can be stored for a long time, and the detection performance does not decline. (3) Due to the excitation in the near-infrared region (980nm), the generation of background fluorescence is avoided. Therefore, it has a good application prospect.

Description

A quantitative detection method for double recognition microRNA

technical field

The invention belongs to the field of biotechnology and clinical medical diagnosis, and relates to a quantitative detection method for double-identification detection of microRNA. Based on upconversion fluorescence energy resonance transfer (UC-LRET) technology combined with different hybridization (hybridization) and ligation (ligation) double recognition detection, it can realize single-step, homogeneous and highly sensitive detection of microRNA. It is mainly used in microRNA expression profile analysis, microRNA clinical diagnosis, microRNA research and detection, and microRNA-related drug research and screening.

Background technique

microRNA (hereinafter referred to as "miRNA") is a kind of non-coding RNA with a length of about 20-24 nucleotides (nt), which can regulate more than half of human genes. [See (a) D.P. Bartel, Cell, 2009, 136, 215-233. (b) S.L. Ameres, M.D. Horwich, J.H. Hung, J. Xu, M. Ghildiyal, Z. Weng and P.D. Zamore, Science, 2010, 328 , 1534-1539.(c) Y.Chen, D.Y.Gao and L.Huang, Adv.Drug Delivery Rev.2015, 81, 128.(d) R.Devulapally, N.M.Sekar, T.V.Sekar, K.Foygel, T.F.Massoud, J.R.K.Willmann and R.Paulmurugan, ACS Nano, 2015, 9, 2290.] The same miRNA has similar regulatory functions in different tissues, organs and different cells, and is conserved. However, different tissues and organs, different cells, and different stages of cell development have different miRNA expression profiles. This cell and "spatial-temporal" specificity makes it possible to be used as a specific expression profile for certain tissues, organs, different cells, and different stages of cell development. Sex molecule sign. In recent years, miRNA is considered to be a new generation of markers and targets for the diagnosis and treatment of certain diseases. [See (a) R. Ma, T. Jiang and X. Kang, J. Exp. Clin. Cancer Res. 2012, 31, 38. (b) Y. Cao, R. A. DePinho, M. Ernst and K. Vousden, Nat. Rev.Cancer, 2011, 11, 749.] miRNA has become a research hotspot in recent years, however, due to the characteristics of very low concentration, easy degradation, short chain length (18-24nt) and strong sequence similarity of miRNA This makes sensitive and specific detection of miRNA very difficult. At present, microarray chip "hybridization" and quantitative real-time polymerase chain reaction (PCR) technology are the two most common techniques for quantitative detection of miRNA. [See A. Git, H. Dvinge, M. Salmon-Divon, M. Osborne, C. Kutter, J. Hadfield, P. Bertone and C. Caldas, RNA, 2010, 16, 991.]. However, the difference in melting point temperature of miRNAs caused by sequence diversity makes hybridization-based techniques difficult to apply to large-scale expression analysis, and PCR-based techniques require multiple procedures such as miRNA labeling and complementary DNA conversion steps, making Detection of miRNA is time-consuming and labor-intensive. [See (a) Z. Gao, H. Deng, W. Shen and Y. Ren, Anal. Chem. 2013, 85, 1624-1630. (b) Y. Ren, H. Deng, W. Shen and Z. Gao , Anal.Chem.2013, 85, 4784-4789.] Therefore, the development of rapid, simple and sensitive biological detection miRNA technology is of great significance in biological research and clinical diagnosis.

Contents of the invention

In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a method for highly sensitive detection of miRNA based on up-conversion fluorescence resonance energy transfer (UC-LRET) technology, which can provide "background-free" homogeneous detection in a biological environment , and through different hybridization "hybridization" and fusion "ligation steps" double recognition steps, the specificity of detection is greatly improved.

The present invention is composed of a signal generation unit and a recognition unit, and the oligonucleotide labeled with upconversion nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) is used as a fluorescence donor, and the oligonucleotide coupled with dye Cy3 As a fluorescent acceptor, the donor-acceptor together constitute a pair of up-converting fluorescent energy resonance transfer oligonucleotides as a signal generation unit; the recognition unit includes two sequences that specifically recognize the target miRNA. Under the excitation of near-infrared light (980nm), an upconversion fluorescence resonance energy transfer signal is generated, which can realize high sensitivity and high selectivity detection of miRNA.

The technical solution adopted:

An upconversion nanoparticle (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B) for quantitative detection of miRNA, wherein the oligonucleotide sequence starts from the 5' end It consists of a specific sequence complementary to the recognition unit, a sequence controlling the length of the oligonucleotide, and a modified C7 amino sequence at the 3' end.

A dye molecule Cy3-labeled oligonucleotide (LRET-A) for quantitative detection of miRNA, wherein the oligonucleotide sequence is marked with a Cy3 control oligonucleotide length sequence from the 5' end, and The specific sequence of the complementary pairing of the recognition unit and the C6 modification at the 3' end.

A recognition unit oligonucleotide (RD-REG-D) for the quantitative detection of miRNA, wherein the oligonucleotide sequence starts from the 5' end of _PO4 modification and the specific sequence that is complementary to the target miRNA, The specific sequence and 3' end composition that are complementary to the oligonucleotide (LRET-B).

A recognition unit oligonucleotide (RA-REG-A) for quantitative detection of miRNA, wherein the oligonucleotide sequence starts from the 5' end and is complementary to the oligonucleotide (LRET-A). It consists of a sex sequence, a specific sequence that is complementary to the target miRNA, and a 3' end modified with an -OH group.

A method for highly sensitive detection of miRNA based on upconversion fluorescence energy resonance transfer (UC-LRET) technology, carried out according to the following steps:

(1) Up-converting nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B): 1-3 ml of carboxylated up-converting nanoparticles UCNPs solution with a concentration of 0.2-2 mg/ml Add 2-6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-4 mg of N-hydroxysuccinimide (NHS) to the Shake for 60 minutes, then add 50-150 μL of 0.5-2 μM oligonucleotide (LRET-B) solution into the above solution, and react at room temperature for 12-24 hours. After the reaction was completed, the product was ultrafiltered and centrifuged, and the product was stored in BSA buffer (pH 7.4) containing 0.1% bovine serum albumin.

(2) Quantitative detection based on dual-recognition microRNA: 5-15nM up-conversion nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B), 5-15nM dye molecule Cy3 labeling oligonucleotide (LRET-A), 5~15nM two double recognition unit oligonucleotides (RA-REG-A), (RD-REG-D), 0.02~0.12CEU RNA ligase and a certain concentration of The target miRNA was mixed in 100 μL of miRNA buffer, and the composition of miRNA buffer was (25mM HEPES buffer, 0.4mMATP, 50mM NaCl, 2mM MgCl ₂ , 100μg/ml single stranded salmon sperm DNA, pH7.4+0.1%BSA). The mixed samples were incubated at 20-40°C for 60-120 min, and finally carried out quantitative analysis of up-conversion fluorescence. The excitation light is excited by a 980nm laser light source, and the power is adjusted between 0 and 3W.

Compared with the existing "microarray hybridization method" and "quantitative PCR amplification method", the method for highly sensitive detection of miRNA based on up-conversion fluorescence energy resonance transfer (UC-LRET) technology provided by the present invention has the following advantages:

(1) There are few operation steps, which belong to the "one-step" detection. However, the "microarray hybridization method" and "quantitative PCR amplification method" require hybridization reaction, amplification, staining and staining, and there are many analysis steps, which are prone to errors in the process.

(2) It has good stability, can be stored for a long time, and the detection performance does not decline.

(3) Due to the excitation in the near-infrared region (980nm), the generation of background fluorescence is avoided.

In the process of completing the invention, miRNA-21, miRNA-125a, and miRNA-125b were selected as detection targets, and various performance indicators of the method for detecting miRNAs based on up-conversion fluorescence energy resonance transfer (UC-LRET) were carried out. repeated tests. The results show that the linear range of miRNAs detection is 200pM-1.4nM, the repeatability of seven measurements is 3.9%, and the recovery rate is 93-105% in the detection of miRNA-21 in tumor cell Hela extract.

Description of drawings

Figure 1 is a schematic diagram of the highly sensitive detection of miRNAs based on upconversion fluorescence resonance energy transfer (UC-LRET) technology.

Figure 2 is a transmission electron microscope image A and a high-resolution transmission electron microscope image B of upconversion nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ );

Figure 3 (A) is the fluorescence spectrum and (B) the linear relationship curve for measuring miRNA-21.

detailed description

Below by embodiment form, above-mentioned content of the present invention is described in further detail again, but this should not be interpreted as the scope of the above-mentioned theme of the present invention being limited to following examples, all technologies realized based on the above-mentioned content of the present invention all belong to this invention the scope of the invention.

The present invention will be further described in detail through specific embodiments below in conjunction with the accompanying drawings.

Example 1: Upconverting nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B)

Experimental materials: bovine serum albumin, batch number 140318, Shanghai Aladdin Reagent Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), batch number 090M14531V, Shanghai Aladdin Reagent Co., Ltd. Latin Reagent Co., Ltd.; succinimide (NHS), lot number MKBG7914V, Shanghai Aladdin Reagent Co., Ltd.; ethanol, lot number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. The oligonucleotide sequence (such as 5'-TTGTGTTCCGATAGGCT-AAAAAAAA 3' C7-NH2) starts from the 5' end to be a specific sequence complementary to the recognition unit, a sequence to control the length of the oligonucleotide, and a modified C7 amino group at the 3' end The sequence composition, in which TTGTGTTCCGATAGGCT is a specific sequence complementary to the recognition unit, varies with the detected miRNA. The oligonucleotide sequence is:

UC-LRET-B: 5'-TTGTGTTCCGATAGGCT-AAAAAAAA 3'C7- _NH2 ;

UC-LRET-A: Cy3-5'AAAAAAAA-CGATCAGTCAGGCAA-3'C6;

the adapter oligos (for miRNA-21):

3'TCCCCAACACAAGGCTATCCGA-ATCGAATAGTCT-5'-PO ₄

3'OH-GACTACAACT-GCTAGTCAGTCCGTTTCGCC5'

(for miRNA-125a):

3'TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5'-PO ₄

3'OH-AAATTGGACACT-GCTAGTCAGTCCGTTTCGCC5'

(for miRNA-125b):

3'TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5'-PO ₄

3'OH-ATTGAACACT-GCTAGTCAGTCCGTTTCGCC5'

miRNA-125a: 5'-UCCCUGAGACCCUUUAACCUGUGA-3'

miRNA-125b: 5'-UCCCUGAGACCCUAACUUGUGA-3'

miRNA-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3

Experimental instruments and equipment: KQ-500DE ultrasonic cleaner, DHG-9143BS electric blast drying oven, AY120 electronic analytical balance, TGL-16C centrifuge.

Experimental procedure: Add 2mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1mg N-hydroxysuccinimide (NHS) was shaken at room temperature for 60 minutes, then 100 μL of 1 μM oligonucleotide (LRET-B) solution was added to the above solution, and reacted at room temperature for 18 hours. The oligonucleotide sequence (such as 5'-TTGTGTTCCGATAGGCT-AAAAAAAA 3'C7-NH ₂ ) starts from the 5' end to be a specific sequence complementary to the recognition unit, a sequence to control the length of the oligonucleotide, and a modified C7 at the 3' end Amino sequence composition, wherein TTGTGTTCCGATAGGCT is a specific sequence complementary to the recognition unit. After the reaction was completed, the product was ultrafiltered and centrifuged, and the product was stored in BSA buffer (pH 7.4) containing 0.1% bovine serum albumin. The labeled up-conversion nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) were detected by transmission electron microscopy and high-resolution transmission electron microscopy. as shown in picture 2.

Example 2: Quantitative detection of miRNA-21 based on dual recognition microRNAs

Experimental material: ethanol, batch number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. HEPES buffer, batch number: E607018, purchased from Shanghai Sangon Bioreagent Co., Ltd.; 5′ adenosine triphosphate disodium salt trihydrate (ATP), batch number: A600020, purchased from Shanghai Sangon Bioreagent Co., Ltd.; single stranded salmon sperm DNA, lot number: C640031, purchased from Shanghai Sangon Bioreagent Co., Ltd.; RNA ligase, lot number: B101801, purchased from Shanghai Sangon Bioreagent Co., Ltd.

The oligonucleotide sequence is:

UC-LRET-B: 5'-TTGTGTTCCGATAGGCT-AAAAAAAA 3'C7- _NH2 ;

UC-LRET-A: Cy3-5'AAAAAAAA-CGATCAGTCAGGCAA-3'C6;

the adapter oligos (for miRNA-21):

(RD-REG-A): 3'TCCCCAACACAAGGCTATCCGA-ATCGAATAGTCT-5'- _PO4

(RD-REG-D): 3’OH-GACTACAACT-GCTAGTCAGTCCGTTTCGCC5’

miRNA-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3

Experimental instruments and equipment: KQ-500DE ultrasonic cleaner, DHG-9143BS electric blast drying oven, AY120 electronic analytical balance, TGL-16C centrifuge.

Experimental procedure: 5nM up-conversion nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B), 5nM dye molecule Cy3-labeled oligonucleotide (LRET-A), 5nM two A double recognition unit oligonucleotide (RA-REG-A), (RD-REG-D), 0.10CEU RNA ligase and a certain concentration of target miRNA-21 were mixed in 100 μL miRNA buffer, and the composition of miRNA buffer was (25 mM HEPES buffer, 0.4 mM ATP, 50 mM NaCl, 2 mM MgCl ₂ , 100 μg/ml singlestranded salmon sperm DNA, pH7.4+0.1% BSA). The mixed samples were incubated at 25°C for 90 min, and finally the up-conversion fluorescence quantitative analysis was performed. The excitation light is excited by a 980nm laser light source, and the power is adjusted between 0 and 3W. (The measured up-conversion fluorescence spectrum and the calibration curve are shown in Figure 3)

Example 3: Quantitative detection of miRNA-125a based on dual recognition microRNAs

Experimental material: ethanol, batch number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. HEPES buffer, batch number: E607018, purchased from Shanghai Sangon Bioreagent Co., Ltd.; 5′-adenosine triphosphate disodium salt trihydrate (ATP), batch number: A600020, purchased from Shanghai Sangon Bioreagent Co., Ltd.; single stranded salmon Sperm DNA, batch number: C640031, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; RNA ligase, batch number: B101801, purchased from Shanghai Sangon Biological Reagent Co., Ltd.;

The oligonucleotide sequence is:

UC-LRET-B: 5'-TTGTGTTCCGATAGGCT-AAAAAAAA 3'C7- _NH2 ;

UC-LRET-A: Cy3-5'AAAAAAAA-CGATCAGTCAGGCAA-3'C6;

the adapter oligos (for miRNA-125a):

(RD-REG-A): 3'TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5'- _PO4

(RD-REG-D): 3’OH-AAATTGGACACT-GCTAGTCAGTCCGTTTCGCC 5’

miRNA-125a: 5'-UCCCUGAGACCCUUUAACCUGUGA-3

Experimental instruments and equipment: KQ-500DE ultrasonic cleaner, DHG-9143BS electric blast drying oven, AY120 electronic analytical balance, TGL-16C centrifuge.

Experimental process: 5nM up-converting nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B), 5nM dye molecule Cy3-labeled oligonucleotide (LRET-A), 5nM two A double recognition unit oligonucleotide (RA-REG-A), (RD-REG-D), 0.10CEU RNA ligase and a certain concentration of target miRNA-125a were mixed in 100 μL miRNA buffer, and the miRNA buffer consisted of (25 mM HEPES buffer, 0.4 mM ATP, 50 mM NaCl, 2 mM MgCl ₂ , 100 μg/ml single stranded salmon sperm DNA, pH7.4+0.1% BSA). The mixed samples were incubated at 25°C for 90 min, and finally the up-conversion fluorescence quantitative analysis was performed. The excitation light is excited by a 980nm laser light source, and the power is adjusted between 0 and 3W.

Example 4: Quantitative detection of miRNA-125b based on dual recognition microRNAs

Experimental material: ethanol, batch number: 14021710247, purchased from Nanjing Chemical Reagent Co., Ltd. HEPES buffer, batch number: E607018, purchased from Shanghai Sangon Bioreagent Co., Ltd.; 5′-adenosine triphosphate disodium salt trihydrate (ATP), batch number: A600020, purchased from Shanghai Sangon Bioreagent Co., Ltd.; single stranded salmon Sperm DNA, batch number: C640031, purchased from Shanghai Sangon Biological Reagent Co., Ltd.; RNA ligase, batch number: B101801, purchased from Shanghai Sangon Biological Reagent Co., Ltd.;

The oligonucleotide sequence is:

UC-LRET-B: 5'-TTGTGTTCCGATAGGCT-AAAAAAAA 3'C7- _NH2 ;

UC-LRET-A: Cy3-5'AAAAAAAA-CGATCAGTCAGGCAA-3'C6;

the adapter oligos (for miRNA-125b):

(RD-REG-A): 3'TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5'- _PO4

(RD-REG-D): 3’OH-ATTGAACACT-GCTAGTCAGTCCGTTTCGCC5’

miRNA-125b: 5'-UCCCUGAGACCCUAACUUGUGA-3'

Experimental instruments and equipment: KQ-500DE ultrasonic cleaner, DHG-9143BS electric blast drying oven, AY120 electronic analytical balance, TGL-16C centrifuge.

Experimental process: 8nM up-converting nanoparticles (NaYF ₄ : Er ³⁺ , Yb ³⁺ ) labeled oligonucleotide (LRET-B), 8nM dye molecule Cy3-labeled oligonucleotide (LRET-A), 8nM two A double recognition unit oligonucleotide (RA-REG-A), (RD-REG-D), 0.10CEU RNA ligase and a certain concentration of target miRNA-125b were mixed in 100 μL miRNA buffer, and the miRNA buffer consisted of (25mM HEPES buffer, 0.4mM ATP, 50mM NaCl, 2mM MgCl2, 100μg/ml single stranded salmon sperm DNA, pH7.4+0.1%BSA). The mixed samples were incubated at 30°C for 90 min, and finally the up-conversion fluorescence quantitative analysis was performed. The excitation light is excited by a 980nm laser light source, and the power is adjusted between 0 and 3W.

The resulting quantitative relationship graph is shown in Figure 3. The results showed that the linear range of miRNAs detection was 0.2pM～1.4nM, and the repeatability of 7 measurements was 3.9%.

The above examples are only used to illustrate preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations, within the scope of knowledge possessed by those of ordinary skill in the art, any modifications made within the spirit and principles of the invention , equivalent replacements and improvements, etc., all of which should be within the scope of the technical solution claimed in the present invention.

Claims (2)

1. the quantitative detecting method of a kind of couple of recognition detection microRNA, it is characterised in that carry out according to the following steps：

(1), upper conversion nano granule (NaYF₄：Er³⁺, Yb³⁺) labeled oligonucleotide (LRET-B)：1～3ml concentration be 0.2～ 2mg/ml carboxylated upper conversion nano granule (NaYF₄：Er³⁺, Yb³⁺) add 1- (3- dimethylamino-propyls) -3- second in solution The N-hydroxy-succinamide (NHS) of base carbodiimide hydrochloride (EDC) 2～6mg and 1～4mg, vibrates 60 points at room temperature Clock, then adds 0.5～2 μM of 50～150 μ L of oligonucleotide (LRET-B) solution in above-mentioned solution, reacts at room temperature 12～24 hours.After completion of the reaction, ultrafiltration centrifugation, product are stored in containing in 0.1% bovine serum albumin BSA buffer (pH7.4)。

(2), the detection by quantitative based on double identification microRNA：The upper conversion nano granule (NaYF of 5～15nM₄：Er³⁺, Yb³⁺) labelling Oligonucleotide (LRET-B), the oligonucleotide (LRET-A) of 5～15nM dye molecule Cy3 labellings, the double identifications of two kinds of 5～15nM Unit oligonucleotide (RA-REG-A), (RD-REG-D), 0.02～0.12CEU RNA ligases and certain density target MiRNA is blended in 100 μ L miRNA buffer, miRNA buffer consist of (25mM HEPES buffer solutions, 0.4mM ATP, 50mM NaCl, 2mM MgCl₂, 100 μ g/ml single stranded salmon sperm DNA, pH7.4+0.1% BSA).Biased sample is incubated 60～120min at 20～40 DEG C, finally carries out up-conversion fluorescence quantitative analyses.

2. the quantitative detecting method of a kind of couple of recognition detection microRNA according to claim 1, it is characterised in that：

(1) with upper conversion nano granule (NaYF₄：Er³⁺, Yb³⁺) labelling oligonucleotide (LRET-B), its sequence be 5 '- TTGTGTTCCGATAGGCT-AAAAAAAA3’C7-NH₂, wherein 3 ' end C7-NH₂Modification, the sequence such as SEQ ID NO：1 institute Show.

(2) oligonucleotide (LRET-A) of dye molecule Cy3 labellings, its sequence are Cy3-5 ' AAAAAAAA- CGATCAGTCAGGCAA-3 ' C6, wherein 5 ' end Cy3 modifications, 3 ' end C6 modifications, the sequence such as SEQ ID NO：Shown in 2.

(3) double recognition unit oligonucleotide (RA-REG-A), its sequence is：

To detect miRNA-21：5’PO₄- TCTGATAAGCTA-AGCCTATCGGAACACAACCCCT-3 ', wherein 5 ' end PO₄ Modification, the sequence such as SEQ ID NO：Shown in 3.

To detect miRNA-125a：5’PO₄- GGGTCTCAGGGA-AGCCTATCGGAACACAACCCCT-3 ', wherein 5 ' ends PO₄Modification, the sequence such as SEQ ID NO：Shown in 4.

To detect miRNA-125b：5’PO₄-GGGTCTCAGGGA-AGCCTATCGGAACACAACCCCT-3’；Wherein 5 ' ends PO₄Modification, the sequence such as SEQ ID NO：Shown in 5.

(4) double recognition unit oligonucleotide (RA-REG-D), its sequence is：

To detect miRNA-21：5’-CCGCTTTGCCTGACTGATCG-TCAACATCAG-OH3’；Wherein 3 ' end OH modifications, The sequence such as SEQ ID NO：Shown in 6.

To detect miRNA-125a：5’-CCGCTTTGCCTGACTGATCG-TCACAGGTTAAA-OH3’；Wherein 3 ' end OH are repaiied Decorations, the sequence such as SEQ ID NO：Shown in 7.

To detect miRNA-125b：5’-CCGCTTTGCCTGACTGATCG-TCACAAGTTA-OH3’；Wherein 3 ' end OH are repaiied Decorations, the sequence such as SEQ ID NO：Shown in 8.